Patients with diabetes mellitus exhibit post-prandial hyperglycemia, systemic oxidative stress, impaired endothelium-dependent, nitric oxide (NO)-mediated coronary artery dilatation, and an increased incidence of coronary events. Whether hyperglycemia causally mediates these associations is unknown. To test the hypothesis that post-prandial hyperglycemia acutely impairs coronary endothelial function in humans, we compared the ability of the endothelium-dependent vasodilator acetylcholine to increase conduit coronary diameter (the macrovascular response) and coronary blood flow velocity (the microvascular response) in 12 cardiac transplant recipients without diabetes before and after raising blood glucose from 6.7±1.3 mmol L^-1 (121±24 mg dl^-1) to 17.8±1.5 mmol L^-1 (321±27 mg dl^-1) for one hour. Hyperglycemia acutely doubled circulating levels of the oxidation product malondialdehyde, indicating systemic oxidative stress, but did not affect acetylcholine's ability to dilate conduit coronary segments or accelerate coronary blood flow. We conclude the oxidative stress associated with a single acute episode of hyperglycemia affects neither acetylcholine-mediated coronary endothelial NO release, nor the subsequent bioavailability, metabolism or action of NO within the coronary circulation of cardiac transplant recipients. These observations imply that the relationship between hyperglycemia, oxidative stress and coronary endothelial dysfunction is presumably mediated by mechanisms operating over longer periods of time.